An in vitro fluorescence based study of initiation of RNA synthesis by influenza B polymerase.
Nucleic Acids Res
; 45(6): 3353-3368, 2017 04 07.
Article
em En
| MEDLINE
| ID: mdl-28126917
ABSTRACT
Influenza polymerase replicates, via a complementary RNA intermediate (cRNA), and transcribes the eight viral RNA (vRNA) genome segments. To initiate RNA synthesis it is bound to the conserved 5Î and 3Î extremities of the vRNA or cRNA (the 'promoter'). 5Î-3Î base-pairing in the distal promoter region is essential to position the template RNA at the polymerase active site, as shown by a new crystal structure with the 3Î end threading through the template entry tunnel. We develop fluorescence polarization assays to quantify initiation of cap-primed (transcription) or unprimed (replication) RNA synthesis by recombinant influenza B polymerase bound to the vRNA or cRNA promoter. The rate-limiting step is formation of a primed initiation complex with minimally ApG required to stabilize the 3Î end of the template within the active-site. Polymerase bound to the vRNA promoter initiates RNA synthesis terminally, while the cRNA promoter directs internal initiation at a significantly lower rate. Progression to elongation requires breaking the promoter 5Î-3Î base-pairing region and favourable compensation by the emerging template-product base-pairs. The RNA synthesis assay is adaptable to high-throughput screening for polymerase inhibitors. In a pilot study, we find that initiation at the cRNA promoter is unusually susceptible to inhibition by 2ÎF-2ÎdNTPs.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Vírus da Influenza B
/
Proteínas Virais
/
RNA Polimerases Dirigidas por DNA
/
RNA Viral
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article